One thing you want to be cautious about when using that much template in a
reaction is salt carryover from the prep. We have used the qiagen
nucleotide removal kit with good success when encountering this problem.
Jeff Fairman
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Jeff Fairman, Ph.D.
Senior Scientist, Pharmacogenomics Research
Clingenix, Inc.
871 Industrial Road, Suite J
San Carlos, CA 94070
(650) 598-7645 (office)
(650) 598-7641 (fax)
jfairman at clingenix.com
visit: http://thelabrat.netfirms.com - By Scientists... For Scientists.
jkcarson at netspace.net.au wrote in message
<383bddbc.49782822 at news.netspace.net.au>...
>We have been using the guanidinium thiocyanate - diatom method for
>extracting bacterial DNA and has been working well. The extracted DNA
>is used for a 16S rRNA amplification. Our PCR reaction volume is 20ul
>and we use a 1ul template volume. We have modified the PCR format so
>that we can use 9ul of template in the 20ul PCR format - a strategy to
>increase sensitivity. We validated the modification using DNA purified
>by phenol-chloroform.
>If we use a 9ul volume of template DNA prepared using the guanidinium
>method we do not get any PCR product but if we use a 1ul volume as
>template the PCR works. We have concluded that an inhibitor is
>co-extracting. BSA does not neutralise the effect.
>Does anyone have any suggestions on how to neutralise the PCR
>inhibitor? Has anyone tried GeneReleaser for post-extraction cleanup
>or used alpha-casein to neutralise PCR inhibitors. For various reasons
>we are locked in to the guanidinium-diatom method of DNA extraction.
>Thanks
>Jeremy Carson
>Dept Primary Industry
>Tasmania, Australia
>jeremy.carson at dpiwe.tas.gov.au